The present study was undertaken to evaluate serum levels of pro-inflammatory cytokines in Tunisian older adults and to examine the relationships between inflammatory marker levels, geriatric, and biochemical parameters. A cross-sectional study was conducted in a population of Tunisian older adults (N = 141, aged 65 and over). Patients were recruited from the Department of Internal Medicine, Fattouma Bourguiba University Hospital (Monastir, Tunisia) and from a nursing home (Sousse, Tunisia). Comprehensive geriatric assessment, history taking and examination including functional and nutritional assessment were done for each participant. Enzyme-linked immunosorbent assay (ELISA) test was used to measure serum cytokine (TNF-α, IL-8, IL-6) levels. The modified Short Emergency Geriatric Assessment score (SEGAm) were used to classify patients as 51 very-frail, 40 frail, and 50 non-frail. The age of the participants (80 men, 61 women) ranged from 65 to 97 years. Serum levels of TNF-α, IL-8 and C-reactive protein (CRP) were significantly higher in very-frail participants compared to frail and non-frail ones. However, no significant differences in IL-6 levels were detected among frailty groups. After adjustment for age, CRP and IL-8 levels remained significantly associated with frailty. Analysis of the receiver operating characteristic (ROC) curve corresponding to IL-8 showed an area under the curve of 0.7 (p = 0.003; 95% CI [0.58–0.81]) and a predictive threshold of 5.27 pg/ml. Positive correlations were found between frailty score, IL-6, and IL-8 levels. In addition, a significant positive correlation was observed between IL-8 levels and Timed Up and Go test results. However, a negative correlation was observed between Mini Nutritional Assessment Short-Form score, IL-6 and CRP levels, as well as between Activities of Daily Living score and serum levels of TNF-α, IL-6, and CRP. In conclusion, the key findings of this study collectively support a role of pro-inflammatory cytokines, TNF-α, CRP, and especially IL-8 in the development of frailty in older adults.